IGEM:IMPERIAL/2007/Projects/Hrp System/Characterisation BBa F2620

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Characterisation of BBa_F2620

  • To date the best characterisation of a part has been BBa_F2620. This is a LuxR promoter

This is a summary of the approach they used in order to help us design a protocols to characterise the Hrp system.

1 . Transfer Function

  • We can measure the transfer function indirectly using GFP and measuring fluorescence, so see how the output will change with a range of inputs.
    • General protocol- look at fluorescence in terms of molecules of GFP per CFU-1 s-1 (CFU-Colony forming units) at varying concentrations at varying time points. Repeated experiments on 3 days for 9 colonies on 1 plate.
    • The key ideas to get form this - how to count the cell number, how to work on the fluorescence in terms of molecules of GFP and how this is then used to work out the mean GFP synthesis rates per cell.
    • The reason why this is useful is that were turning our information into an approximation of proteins synthesized. This approximation is more useful to us when building a genetic circuit however, it is still far off being able to produce an accurate approximation of PoPs.
    • From doing these sets of experiments to obtain a graph of then GFP per CFU-1 s-1 vs inducer input concentration from this we can obtain the input swing, output swing and the switch point.
      • Input Swing- The range at which the inputs result in an output of 5%-95%
      • The switch point is the input level at which the output is 50% of maximum outputs.
      • The Output swing is the maximum level of output (CHECK)
    • On ours our input is going to be what ever inducible promoter needs to be investigated, this gives a set number of PoPS.

  • In our device look at the possibility of measuring both inputs and outputs in terms of protein molecules produced. This will give a more generic characterisation because we have separated our characterisation of the device from the inducible promoter used.
  • The key measurements are:
  • Measure absorbance at 500nm of wells
    • Used to calculate cell density
  • Measure fluorescence emitted
    • Representation of total GFP of well
  • These measurements are made at time intervals of 2 minutes 21 seconds
  • In addition, they are measured over a range of inducer concentration.
    • Varying inducer concentration will be change the PoPs into the device

Data analysis
  1. Combine information in 2,3,4 to give a unit of GFP molecules cfu -1 s-1
  2. To do this need to create two data calibration curves, first for colony forming units and secondly for linking fluorescence to GFP molecules.
Steady State
  1. Can test the steady state of the host by measuring growth by absorbance and linking this to this to the CFU to absorbance ratio.
  • linking to our system may consider combining these two ideas:

Screening Plasmid Technique Where we could use RFP as our input and GFP as our output

2. Specificity

  • The specificity of the promoter was tested by testing the response to a variety to a variety of inducer analogys.
  • A serious of experiments were carried out for a 7 anaogues. The protocol used was the same as the one carried out for the transfer function.
  • However, in this protocol there were only two cultures used, one containing a functional GFP and one containnig a mutant verision.
  • The key measurements,
  1. Measure absorbance at 500nm of wells
  2. Measure fluorescence emitted
  • These measurements are made every 2 minutes and 21 seconds and measured for a range of different anaolgues at varying concentrations (8 in total).

3. Response Times

  • To measure the response time then AHL is added at a concentration that will cause the maxium output. This will be determined by the transfer function experiements.
  • Two cultures are used in these experiments, one contains a mutant GFP to enable the back ground fluorescence to be determined and one culture to test the reponse time.
  • To the normal GFP culture, normal GFP the absorbance and fluorescence are measured. Using the same calibration curves this information is converted into the GFP molecules synthesis CFU -1 s-1
  • low-high reponse time was then measured for 50% and 90% output.

4. Stability

  • This is a measure of how stable the transfer function of the device is after multply rounds of replication.